Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will focus on several key questions regarding the mechanisms of reversible plasma membrane localization of heterotrimeric (alpha beta gamma) G proteins. G proteins act as molecular switches to relay information from cell surface receptors to appropriate effector proteins. To transmit a signal, G proteins must be localized, at least initially, to the cytoplasmic face of the plasma membrane. G protein alpha subunits (Galpha) are modified by the covalent attachment of the fatty acids myristate and/or palmitate, and gamma subunits of the beta gamma dimers are modified by arnesyl or geranylgeranyl lipid moieties. These attached lipids likely function as hydrophobic anchors to promote binding to cellular membranes; however, additional membrane targeting signals for heterotrimeric G proteins have not been well described. Moreover, when and where inside the cell does the heterotrimer initially form, and what is the cellular pathway used by G proteins to arrive at the plasma membrane, are critical questions that remain unanswered. Once the heterotrimeric G protein is activated at the plasma membrane, Galpha and beta gamma dissociate, and Galpha can undergo rapid depalmitoylation. For one Galpha alphas, receptor activation also promotes its translocation off the plasma membrane and into the cytoplasm of the cell. The underlying mechanisms and cellular pathways of this G protein trafficking are also poorly understood. Thus, the major objectives of this proposal are 1) Define the role of Get in plasma membrane targeting of Gbata gamma; 2) Define the cellular pathway used by Galpha and Gbeta gamma in trafficking to the plasma membrane after synthesis; 3) Define the role of an N-terminal polybasic cluster in membrane targeting of Galpha; and 4) Define mechanisms of activation-induced redistribution of Galphas. These objectives focus on distinct yet highly related questions of the cell biology of G proteins. This research will utilize cultured mammalian cells as model systems and will employ a number of techniques, including immunofluorescence microscopy, fluorescence microscopy of live cells, subcellular fractionation, and numerous biochemical assays to define structure -function relationships in terms of mechanisms of reversible membrane targeting of G proteins.